The present invention relates to an automatic enzyme immonoassay apparatus capable of performing enzyme immunoassay automatically, and more particularly to an apparatus for measuring the amount of antigens, haptens or antibodies present in serum.
Enzyme immunoassay is an assay for measuring the amount of an antigen, a hapten or an antibody contained in serum, utilizing an antigen-antibody reaction with the aid of an enzyme. For a more specific explanation of enzyme immunoassay, an example of enzyme immunoassay, the so-called sandwich method, will now be explained.
In the sandwich method, a predetermined amount of an antibody is caused to be deposited on a carrier, for instance, on the surface of a plastic bead, and is made insoluble by converting the antibody to a solid phase fixed to the surface of the carrier.
To the antibody bearing bead is added a predetermined amount of a test solution containing an antigen, the amount of which is to be measured (hereinafter referred to as the test antigen). The antigen is combined with the solidified antibody by the antigen-antibody reaction. The test solution is then removed and the bead is cleaned. A predetermined amount of a reaction solution containing a predetermined amount of an enzyme-labelled antibody, for example, a peroxidase-labelled antibody, is added to the bead. Again an antigen-antibody reaction takes place between the enzyme-labelled antibody and the antigen which has been combined with the antibody solidified and deposited on the bead. The enzyme-labelled antibody which has not combined with the antigen is eliminated, and the activity of the enzyme-labelled antibody which has combined with the antigen is measured, whereby the amount of the test antigen is measured.
In this case, when measuring the activity of peroxidase of the enzyme-labelled antibody which has combined with the solidified antigen, hydrogen peroxide and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) are, for example, employed as a substrate for the enzyme. The bead is washed and unreacted excess peroxidase-labelled antibody is eliminated from the bead. The bead is then immersed in the enzyme substrate solution, and the enzyme substrate solution is colored by the enzyme. The absorbance of the enzyme substrate solution is determined at 405 nm by a spectrophotometer.
In the meantime, reference solutions each containing a known amount of the antigen are prepared and the absorbance of each reference solution is measured, following the steps described above. Thus, a calibration curve is obtained by plotting the amount of the antigen contained in each solution and the absorbance thereof. This calibration curve shows the relationship between the concentration of the antigen in each solution and the absorbance of the solution. By use of that calibration curve, the amount of the antigen contained in the test solution can be determined.
Conventionally, each step in the enzyme immunoassay is conducted manually and, therefore, it takes much time and the results obtained by the manual enzyme immunoassay are not always accurate.